Identification of key amino acids in the gastrin-releasing peptide receptor (GRPR) responsible for high affinity binding of gastrin-releasing peptide (GRP)

The bombesin (Bn) receptor family includes the gastrin-releasing peptide (GRPR) and neuromedin B (NMBR) receptors, Bn receptor subtype 3 (BRS-3) and Bn receptor subtype 4 (BB(4)). They share 50% homology, yet their affinities for gastrin-releasing peptide (GRP) differ. The determinants of GRP high affinity for GRPR and BB(4), and low affinity for BRS-3 are largely unknown. To address this question we made an analysis of structural homologies in Bn receptor members correlated with their affinities for GRP to develop criteria to identify amino acids important for GRP selectivity. Fourteen differences were identified and each was mutated singly in GRPR to that found in hBRS-3. Eleven mutants had a loss of GRP affinity. Furthermore, three of four amino acids in the GRPR selected used a similar approach and previously reported to be important for high affinity Bn binding, were important for GRP affinity. Some GRPR mutants containing combinations of these mutations had greater decreases in GRP affinity than any single mutation. Particularly important for GRP selectivity were K101, Q121, A198, P199, S293, R288, T297 in GRPR. These results were confirmed by making the reverse mutations in BRS-3 to make GRP gain of affinity mutants. Modeling studies demonstrated a number of the important amino acids had side-chains oriented inward and within 6A of the binding pocket. These results demonstrated this approach could identify amino acids needed for GRP affinity and complemented results from chimera/mutagenesis studies by identifying which differences in the extracellular domains of Bn receptors were important for GRP affinity.     

A synthetic glycine-extended bombesin analogue interacts with the GRP/bombesin receptor

alpha-amidation of a peptide (which takes place from a glycine-extended precursor) is required to produce biologically active amidated hormones, such as gastrin-releasing peptide (GRP)/Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH(2) (bombesin). It was shown that glycine-extended gastrin mediates mitogenic effects on various cell lines by interacting with a specific receptor, different from the classical CCK(1) or CCK(2) receptors. On the basis of this observation, we have extended the concept of obtaining active glycine-extended forms of others amidated peptides to produce new active analogues. In this study, we have tested the biological behaviour of a synthetic analogue of the glycine-extended bombesin (para-hydroxy-phenyl-propionyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-Gly-OH or JMV-1458) on various in vitro models. We showed that compound JMV-1458 was able to inhibit specific (3-[125I]iodotyrosyl(15)) GRP ([125I]GRP) binding in rat pancreatic acini and in Swiss 3T3 cells with K(i) values of approximately 10(-8) M. In isolated rat pancreatic acini, we found that JMV-1458 induced inositol phosphates production and amylase secretion in a dose-dependent manner. In Swiss 3T3 cells, the glycine-extended bombesin analogue dose-dependently produced [3H]thymidine incorporation. By using potent GRP/bombesin receptor antagonists, we showed that this synthetic glycine-extended bombesin analogue induces its biological activities via the classical GRP/bombesin receptor.     

Gastrin-releasing peptide receptor in rat brain membranes: specific binding and stimulation of phosphoinositide breakdown

The binding of gastrin-releasing peptide (GRP) to rat brain membranes was characterized. GRP binds specifically to a high affinity site in rat brain membranes, with a Kd equal to 2 nM and Bmax equal to 5 pmol/g wet weight of tissue. The specific binding is saturable, reversible, and dependent on tissue concentration, time of incubation, and the pH of the buffer. Hippocampus, cortex, and striatum contained the highest concentration of high affinity binding sites and the thalamus the lowest. The affinities of GRP, bombesin, and their analogues for the GRP receptor were determined. GRP(14-27) and [Tyr4]bombesin had the greatest affinity, whereas GRP(1-16), which lacks the COOH terminal region, had no affinity for the receptor. GRP, bombesin, and analogues stimulate the breakdown of phosphatidylinositol in rat brain hippocampal minces and potencies correspond to their affinities for the GRP receptor.     

Solid phase synthesis and characterization of two canine gut gastrin-releasing peptides

Two canine gastrin-releasing peptides originally isolated from gut tissue extracts have been synthesized by solid phase methodology and purified by preparative reverse phase high performance liquid chromatography (RP-HPLC). The synthetic gastrin-releasing peptides GRP1-27 and GRP 5-27 were characterized with regard to homogeneity and composition using nine different RP-HPLC systems, mass spectroscopy, amino acid analysis, Edman degradation, methionine oxidation, and peptide mapping with tryptic, Staph. aureus V8 protease and cyanogen bromide cleavage (the latter two systems performed only with GRP 1-27). Although a scarcity of the natural products prevented quantitative biological comparison of the synthetic and natural peptides, they were found to elute identically on RP-HPLC co-chromatography and similar dose dependent biological potencies were observed in canine antral muscle tissue contraction experiments. Indeed, all the peptides containing the bombesin-like car-boxyl terminal decapeptide sequence studied to date have similar biological activities.     

Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal d-Trp or d-Tpi

In an attempt to produce more powerful (effective) bombesin/GRP receptor antagonists, the d forms of Trp or Trp analog (Tpi) were introduced at position 6 in two pseudononapeptides, Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin(6-14) and Leu¹³Ψ(CH₂NH)Phe¹⁴ -bombesin (6-14). These antagonists were tested for their ability to inhibit basal and gastrin releasing peptide (GRP) (14-27)-induced amylase release from rat pancreatic acini in a superfusion assay. They were also assessed for the inhibition of ¹²⁵I-Tyr⁴ -bombesin binding to Swiss 3T3 and small cell lung carcinoma cell line H-345 and the mitogenic response of Swiss 3T3 cells induced by GRP(14-27). The peptides, when given alone, did not stimulate amylase secretion, but were able to inhibit gastrin releasing peptide (14-27)-induced amylase release. All of the antagonists showed strong binding affinities for Swiss 3T3 and H-345 cells and suppressed the GRP(14-27)-induced increase of [³H]thymidine incorporation into DNA of Swiss 3T3 cells at nanomolar concentrations. Antagonist d -Tpi⁶,Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin (6-14)(RC-3095) was slightly more potent in these assays than d -Trp⁶,Leu¹³Ψ (CH₂NH)Leu¹⁴-bombesin (6-14)(RC-3125). Nevertheless, d -Trp⁶ Leu¹³Ψ (CH₂NH)Phe¹⁴-bombesin (6-14) showed the highest binding affinity for Swiss 3T3 and H345 cells and it was the most potent inhibitor of GRP(14-27)-induced amylase secretion. This antagonist RC-3420 was particularly effective in inhibiting the growth of Swiss 3T3 cells, exhibiting an IC₅₀ value less than 1 nm . Our work indicated that the substitution of d -Trp and d -Tpi at position 6 of the pseudononapeptide bombesin analogs (Ψ^13-14), in which the Met¹⁴ residue is replaced by Leu or Phe, results in potent bombesin/GRP antagonists with improved in vivo activity.     

Function of non-visual arrestins in signaling and endocytosis of the gastrin-releasing peptide receptor (GRP receptor)

Little is known about the role of arrestins in gastrointestinal hormone/neurotransmitter receptor endocytosis. With other G protein-coupled receptors, arrestins induce G protein-uncoupling and receptor endocytosis. In this study, we used arrestin wild-type and dominant-negative mutant constructs to analyze the arrestin dependence of endocytosis and desensitization of the gastrin-releasing peptide receptor (GRP-R). Co-expression of the GRP-R with wild-type arrestin2 and arrestin3 increased not only GRP-R endocytosis but also GRP-R desensitization in arrestin-overexpressing cells. Co-expression of the dominant-negative mutants V53D-arrestin2 or V54D-arrestin3 reduced GRP-R endocytosis. Notably, different trafficking routes for agonist-activated GRP-R-arrestin2 and GRP-R-arrestin3 complexes were found. Arrestin3 internalizes with GRP-R to intracellular vesicles, arrestin2 splits from the GRP-R and localizes to the cell membrane. Also, the recycling pathway of the GRP-R was different if co-expressed with arrestin2 or arrestin3. Using different GRP-R mutants, the C-terminus and the 2nd intracellular loop of the GRP-R were found to be important for the GRP-R-arrestin interaction and for the difference in GRP receptor trafficking with the two arrestin subtypes. Our results show that both non-visual arrestins play an important role in GRP-R internalization and desensitization.     

RC-3095, a bombesin/gastrin-releasing peptide receptor antagonist, impairs aversive but not recognition memory in rats

Bombesin and its mammalian equivalent, gastrin-releasing peptide (GRP), stimulate cell proliferation and are involved in the pathogenesis of several types of human cancer. Bombesin-like peptides also display neuroendocrine activities and regulate neural function. In the present study, we evaluated the effects of the bombesin/GRP receptor antagonist (D-Tpi(6), Leu(13) psi[CH(2)NH]-Leu(14)) bombesin-(6-14) (RC-3095), experimental antitumor drug, on memory in rats. Adult female Wistar rats were treated with an intraperitoneal injection of RC-3095 (0.2, 1.0 or 5.0 mg/kg) 30 min before training in either inhibitory avoidance or novel object recognition tasks. Retention test trials were carried out 1.5 (short-term memory) or 24 h (long-term memory) after training. RC-3095 at the doses of 0.2 or 1.0 mg/kg, but not at the dose of 5.0 mg/kg, impaired both short- and long-term inhibitory avoidance retention, but did not affect recognition memory. The memory-impairing effect of RC-3095 could not be attributed to alterations in sensorimotor functions. The results show that the antitumor drug/GRP antagonist RC-3095 impairs formation of aversive memory.     

STUDIES ON PEPTIDES

The heptacosapeptide amide corresponding to the entire amino acid sequence of gastrin-releasing peptide (GRP) was synthesized by assembling seven peptide fragments followed by deprotection with a new reagent system, IM trifluoro-methanesulfonic acid-thioanisole in TFA. The deprotected peptide was purified by ion-exchange chromatography on CM-cellulose followed by partition chromatography on Sephadex G-25. The latter was found to remove effectively the Met (O) derivative of GRP. The highly purified synthetic GRP was active as synthetic bombesin on the molar basis. A new carboxyl-activating reagent, thiazoline-2-thione, was employed for preparation of the necessary fragments.     

The Gastrin-Releasing Peptide Receptor (GRPR) in the Spinal Cord as a Novel Pharmacological Target

Gastrin-releasing peptide (GRP) is a mammalian neuropeptide that acts through the G protein-coupled receptor, GRP receptor (GRPR). Increasing evidence indicates that GRPR-mediated signaling in the central nervous system plays an important role in many physiological processes in mammals. Additionally, we have recently reported that the GRP system within the lumbosacral spinal cord not only controls erection but also triggers ejaculation in male rats. This system of GRP neurons is sexually dimorphic, being prominent in male rats but vestigial or absent in females. It is suggested that the sexually dimorphic GRP/GRPR system in the lumbosacral spinal cord plays a critical role in the regulation of male sexual function. In parallel, it has been reported that the somatosensory GRP/GRPR system in the spinal cord contributes to the regulation of itch specific transmission independently of the pain transmission. Interestingly, these two distinct functions in the same spinal region are both regulated by the neuropeptide, GRP. In this report, we review findings on recently identified GRP/GRPR systems in the spinal cord. These GRP/GRPR systems in the spinal cord provide new insights into pharmacological treatments for psychogenic erectile dysfunction as well as for chronic pruritus.     

Potent pseudopeptide bombesin-like agonists and antagonists

Bombesin-like pseudopeptides have been synthesized, and certain physicochemical properties and biological activities have been examined. Bombesin and the related peptide litorin were modified at positions 13–14 and 8–9, respectively, with ψ[CH₂S] and ψ[CH₂N(CH₃)]. [Phe¹³ψ[CH₂S]Leu¹⁴]bombesin and [Phe⁸ψ[CH₂S]-Leu⁹]litorin bound to the murine pancreatic bombesin gastrin releasing peptide receptor with similar dissociation constants (K_d= 3.9 and 3.4 nM. respectively). Increased potency was achieved by oxidation of the thiomethylene ether to two diastereomeric sulfoxides (isomer I, K_d= 1.6 nM and isomer II, K_d= 0.89nM. Further oxidation to the sulfone decreased potency ([Phe⁸ψ[CH₂SO₂]Leu⁹]litoin, K_d= 9.9nM). All five analogs were receptor antagonists as determined by phosphatidylinositol turnover in murine pancreas. In contrast to these peptide backbone substitutions, a ψCH₂N(CH₃)] at the 8–9 amide bond position resulted in an agonist. The analogs were compared with those of litorin (K_d= 0.1 nM) and [Leu⁹]litorin (K_d= 0.17 nM) by CD and fluorescence spectroscopy. The CD spectra demonstrated ordered conformation for all the peptides in TFE. Different conformations corresponding to agonist and antagonist peptides were suggested by CD. Based on the pH-dependence of the fluorescence spectra of the peptides in a zwitterionic detergent, two titratable groups were identified (pK_a= 6.3 and 8.5). The lower pK_a is found in the agonist analogs but not in the ψ[CH₂S]-containing antagonist.     

Progress in the development of potent bombesin receptor antagonists.

Bombesin and the mammalian-related peptides gastrin-releasing peptide (GRP), GRP and neuromedin B have been shown to have numerous actions in the CNS, gastrointestinal tract and on growth. However, the role of the peptides in various physiological processes has remained unclear because of the lack of potent antagonists. Recent in vitro studies have described four different classes of bombesin receptor antagonist, some of which are active in the nanomolar range and in vivo. Robert Jensen and David Coy describe recent insights into peptide structural determinants of biological activity. Evidence from structure-function studies have resulted in identification of some analogues that function as potent antagonists in all systems examined. Furthermore, various subtypes of bombesin receptors can now be differentiated by these various classes of antagonist.     

Multifunctional targeted therapy system based on 99mTc/177Lu‐labeled gold nanoparticles‐Tat(49–57)‐Lys3‐bombesin internalized in nuclei of prostate cancer cells

Radiolabeled gold nanoparticles may function simultaneously as radiotherapy and thermal ablation systems. The gastrin‐releasing peptide receptor (GRP‐r) is overexpressed in prostate cancer, and Lys³‐bombesin is a peptide that binds with high affinity to the GRP‐r. HIV Tat(49–57) is a cell‐penetrating peptide that reaches the DNA. In cancer cells, ¹⁷⁷Lu shows efficient crossfire effect, whereas ^99mTc that is internalized in the cancer cell nuclei acts as an effective system of targeted radiotherapy because of the biological Auger effect. The aim of this research was to evaluate the in vitro potential of ^99mTc‐labeled and ¹⁷⁷Lu‐labeled gold nanoparticles conjugated to Tat(49–57)‐Lys³‐bombesin peptides (^99mTc/¹⁷⁷Lu‐AuNP‐Tat‐BN) as a plasmonic photothermal therapy and targeted radiotherapy system in PC3 prostate cancer cells. Peptides were conjugated to AuNPs (5 nm) by spontaneous reaction with the thiol group of cysteine (Cys). The effect on PC3 cell viability after laser heating of the AuNP‐Tat‐BN incubated with the cancer cells was conducted using an Nd:YAG laser pulsed for 5 ns at 532 nm (0.65 W/cm²). For the ^99mTc/¹⁷⁷Lu‐AuNP‐Tat‐BN to be obtained, the ¹⁷⁷Lu‐DOTA‐Gly‐Gly‐Cys and ^99mTc‐HYNIC‐octreotide radiopeptides were first prepared and added simultaneously to a solution of AuNP‐Tat‐BN. ^99mTc/¹⁷⁷Lu‐AuNP‐Tat‐BN (20 Bq/cell) was incubated with PC3 cells, and the effect on the cell proliferation was evaluated after 3 days. Fluorescence images of ^99mTc/¹⁷⁷Lu‐AuNP‐Tat‐BN internalized in nuclei of PC3 were also obtained. After laser irradiation, the presence of AuNP‐Tat‐BN caused a significant increase in the temperature of the medium (46.4 vs 39.5 °C of that without AuNP) resulting in a significant decrease in PC3 cell viability down to 1.3%. After treatment with ^99mTc/¹⁷⁷Lu‐AuNP‐Tat‐BN, the PC3 cell proliferation was inhibited. The nanosystem exhibited properties suitable for plasmonic photothermal therapy and targeted radiotherapy in the treatment of prostate cancer.     

Targeted therapy of breast and gynecological cancers with cytotoxic analogues of peptide hormones

Gynecological cancers such as breast, ovarian, and endometrial carcinoma express receptors for luteinizing hormone-releasing hormone (LHRH), bombesin/gastrin-releasing peptide (BN/GRP), and somatostatin (SST). These tumors are therefore suitable candidates for targeted therapy with cytotoxic hybrid molecules consisting of a cytotoxic radical and a peptide hormone analogue as a carrier. These compounds have been shown to be more active and less toxic in vivo than nontargeted chemotherapy in models of various human cancers which express the respective receptors. The current review summarizes experimental and clinical findings with cytotoxic peptide hormone analogues of LHRH (AN-152 [AEZS 108], AN-207), BN/GRP (AN-215), and SST (AN-238) in breast, ovarian, and endometrial cancers.     

Bombesin receptor antagonists. 3. Irreversible alkylating analogues: melphalan derivatives.

Bombesin (BN)-like peptides (such as GRP, gastrin-releasing peptide) are autocrine growth factors for small cell lung carcinoma (SCLC). BN receptor antagonists can therefore find clinical application in the treatment of this highly malignant disease. The present paper deals with a new class of bombesin analogues carrying a nitrogen mustard at their N-terminus. Due to the irreversible binding to the BN receptor(s), these peptides eventually block the mitogenic effects of the natural ligand(s), regardless of their intrinsic "agonistic" or "antagonistic" structures. In Swiss 3T3 fibroblasts they inhibit [125I]GRP binding in the nanomolar/micromolar range. According to their "agonistic" or "antagonistic" structural features, they do or do not induce [3H]thymidine incorporation and p 115 phosphorylation. In competition experiments, alkylating "antagonists" selectively inhibit BN-induced thymidine incorporation either when given simultaneously with or 24 hours before the BN challenge. Alkylating "agonists" display antagonistic effects only in the sequential treatment.     

Characterization of bombesin receptors in peripheral contractile organs.

1 Guinea-pig and rat urinary bladders, rat stomach and the guinea-pig gall bladder, four isolated organs that show high sensitivity to bombesin, were used to characterize bombesin receptors in peripheral organs. 2 The order of potency of agonists was determined with several naturally occurring peptides of the bombesin series, namely bombesin (BBS), litorin (Lit), neuromedin B (NMB), the gastrin-releasing peptide (GRP 18-27), neuromedin C (NMC) and with some bombesin fragments. It was found that bombesin, neuromedin C, litorin and two bombesin fragments, BBS (6-14) and AcBBS (6-14) had similar activities in the four preparations, while neuromedin B and [Phe6]-neuromedin C were more active on the rat urinary bladder than on the other tissues. 3 The order of potency of agonists determined in the rat urinary bladder was as follows: BBS = NMB greater than Lit greater than NMC greater than [Phe6]NMC = GRP and it was found to be different from that observed in the other preparations: BBS greater than GRP = Lit greater than or equal to NMC much greater than NMB greater than [Phe6]NMC, suggesting the existence of two different bombesin receptors, BBS1 and BBS2. 4 This interpretation was convalidated by the finding that bombesin antagonists, namely Ac.GRP(20-26)OCH3 and Ac.GRP(20-26)OC2H5 reduced or blocked the effects of bombesin-related peptides on BBS2 receptor systems while being completely inactive on the rat urinary bladder (BBS1 system).     

Development of insulin resistance and reversal by thiazolidinediones in C2C12 skeletal muscle cells

AIM/HYPOTHESIS: The aim of this study was to develop an insulin-resistant cell culture model in skeletal muscle cell line by chronic presence of insulin in serum-free medium and to determine the effect of thiazolidinediones on insulin signaling. METHODS: We differentiated C2C12 in a combination of serum-free medium in presence or absence of insulin and determined differentiation by creatine kinase activity, myogenin and MyoD expression. The development of insulin resistance was determined by tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1, phosphatidylinositol 3-kinase activity associated with insulin receptor substrate-1 and glucose uptake. We treated the cells with 50 microM of thiazolidinediones to determine the effect on these parameters. RESULTS: C2C12 cells were differentiated normally in the serum-free medium in the absence or presence of insulin. Chronic treatment of insulin resulted in reduced tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1; activation of phosphatidylinositol 3-kinase was impaired and insulin-stimulated glucose uptake was reduced. The treatment of insulin-resistant cells with thiazolidinediones resulted in the enhancement of insulin signaling pathway by increasing tyrosine phosphorylation of insulin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase activity and glucose uptake. CONCLUSION/INTERPRETATION: These results indicate that insulin resistance can be developed in C2C12 skeletal muscle cell line. These findings implicate a direct mechanism of action of thiazolidinediones on skeletal muscle.     

Desensitization of islet cells to bombesin involves both receptor down-modulation and inhibition of receptor function

The neuropeptide bombesin has a powerful but transient stimulatory effect on insulin secretion in the pancreatic islet cell line HIT-T15. We have previously shown that pretreatment of HIT-T15 cells with a saturating concentration of bombesin (100 nM) for 1.5-2 hr abolishes their secretory response to a second challenge with peptide and decreases [125I-Tyr4]bombesin binding by over 90%. In this study we examined the mechanisms involved in desensitization to bombesin. To determine whether receptor modulation was responsible, we compared the effect of bombesin pretreatment on [125I-Tyr4]bombesin binding and on the ability of bombesin to stimulate insulin release. Both effects occurred very rapidly and were maximal by 10 min. Although pretreatment of cells for 90 min with a subsaturating concentration of bombesin did not affect either the ED50 for bombesin-stimulated secretion or the apparent Kd for bombesin binding, it decreased both the maximum secretory response to a subsequent challenge with the peptide and bombesin receptor number. However, the extent of desensitization was greater than the extent of receptor down-regulation at all times examined during pretreatment and recovery. Furthermore, bombesin was 3 times more potent at inducing desensitization (ED50 = 0.35 +/- 0.08 nM) than down-regulation (ED50 = 1.1 +/- 0.4 nM). These results suggest that desensitization was not due solely to a reduction in receptor number. Because bombesin stimulates diacylglycerol production in HIT-T15 cells, we used the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to determine whether protein kinase C also played a role in desensitization to the peptide. Pretreatment of cells with TPA did not affect either [125I-Tyr4]bombesin binding or the dose dependence for bombesin-stimulated hormone release. However, TPA pretreatment did decrease the maximum secretory response to bombesin by 40% and caused a 50% reduction in bombesin-induced accumulation of inositol triphosphates and elevation of intracellular free calcium. Conversely, bombesin pretreatment reduced the secretory response to TPA by 40%. These studies indicate that the mechanism for desensitization to bombesin is a complex process that involves down-regulation of the bombesin receptor, inhibition of intracellular second messenger production, and reduction of protein kinase C-stimulated secretion.     

Synthesis and immunological properties of bombesin analogs

Bombesin (Bn, pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂) is one of the most potent peptides, possessing a variety of physiological and pharmacological functions. We find from CD spectroscopy that the eight C-terminal residues of bombesin [Bn(7-14)NH₂] have an ordered structure, and replacement of His-12 with Pro of Bn(7-14)NH₂ changes the conformation from ordered to a more unordered form. Antibodies to Bn(7-14)NH₂ cross-react to Bn and gastrin releasing peptide (GRP) in a dose-dependent manner. Antibodies to the Pro-analog do not recognize Bn or GRP. Substitution of the C-terminal amide by isopropylamide [Bn(7-14)NHC₃H₇(i))] makes its antibodies more specific to Bn than to GRP. It appears that this region of the peptide is an important antigenic determinant, which makes these antibodies differentiate between BN and GRP.     

Molecular cloning and characterization of avian bombesin-like peptide receptors: new tools for investigating molecular basis for ligand selectivity

(1) Bombesin (BN), originally isolated from amphibians, is structurally related to a family of BN-like peptides found in mammals, which include gastrin-releasing peptide (GRP) and neuromedin B (NMB). These peptides have important effects on secretion, smooth muscle contraction, metabolism and behavior. Here we report cloning and characterization of two subtypes of BN-like peptide receptors in Aves. (2) The amino-acid sequence of chick GRP-R (chGRP-R) is highly identical with mammalian and amphibian GRP-R, and this receptor showed high affinity for GRP, BN and synthetic bombesin agonist, [D-Phe(6), beta-Ala(11), Phe(13), Nle(14)]bombesin(6-14) ([FAFNl]BN(6-14)). The chGRP-R gene was localized to chicken chromosome 1q23distal-q24proximal, where chick homologs of other human X-linked genes have also been mapped. (3) ChBRS-3.5, having sequence similarities to both mammalian bombesin-like peptide receptor subtype-3 and amphibian bombesin-like peptide receptor subtype-4, showed high affinity for [FAFNl]BN(6-14), moderate affinity for BN, but low affinity for both GRP and NMB. (4) Expression of both receptors was detected in brain, but only chGRP-R was expressed in gastrointestinal (GI) tissues. (5) When expressed in Chinese hamster ovary K1 cells, these receptors mediate intracellular calcium mobilization upon agonist stimulation. These results suggest that a novel BN peptide may occur in Aves as an endogenous ligand for chBRS-3.5. (6) The receptor sequences responsible for ligand selectivities were discussed and this knowledge about avian BN-like peptide receptors will help us to understand the molecular basis for agonist sensitivities of BN-like peptide receptors.     

International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states

The mammalian bombesin receptor family comprises three G protein-coupled heptahelical receptors: the neuromedin B (NMB) receptor (BB(1)), the gastrin-releasing peptide (GRP) receptor (BB(2)), and the orphan receptor bombesin receptor subtype 3 (BRS-3) (BB(3)). Each receptor is widely distributed, especially in the gastrointestinal (GI) tract and central nervous system (CNS), and the receptors have a large range of effects in both normal physiology and pathophysiological conditions. The mammalian bombesin peptides, GRP and NMB, demonstrate a broad spectrum of pharmacological/biological responses. GRP stimulates smooth muscle contraction and GI motility, release of numerous GI hormones/neurotransmitters, and secretion and/or hormone release from the pancreas, stomach, colon, and numerous endocrine organs and has potent effects on immune cells, potent growth effects on both normal tissues and tumors, potent CNS effects, including regulation of circadian rhythm, thermoregulation; anxiety/fear responses, food intake, and numerous CNS effects on the GI tract as well as the spinal transmission of chronic pruritus. NMB causes contraction of smooth muscle, has growth effects in various tissues, has CNS effects, including effects on feeding and thermoregulation, regulates thyroid-stimulating hormone release, stimulates various CNS neurons, has behavioral effects, and has effects on spinal sensory transmission. GRP, and to a lesser extent NMB, affects growth and/or differentiation of various human tumors, including colon, prostate, lung, and some gynecologic cancers. Knockout studies show that BB(3) has important effects in energy balance, glucose homeostasis, control of body weight, lung development and response to injury, tumor growth, and perhaps GI motility. This review summarizes advances in our understanding of the biology/pharmacology of these receptors, including their classification, structure, pharmacology, physiology, and role in pathophysiological conditions.